This work was supported by an Ontario HIV Treatment Network Research Operating Grant (ROG-G645) and Canadian Institutes of Health Research New Investigator Award (MSH-113554) to JEL, and University of Toronto Fellowships to HA, FCA, and JDC. The authors would like to thank Marnie Fusco, Dafna Abelson and Dr. Erica Ollmann Saphire at The Scripps Research Institute (La Jolla, CA) for providing cells, ebolavirus GP expression vector and general advice.

1. Preparation Work - Constructs and Cell Cultures
Before starting the protocol, the gene of interest should be codon-optimized for expression in mammalian cells, and cloned into an appropriate expression vector using standard molecular biology techniques. In order to ensure the highest chance for successful expression, multiple variants of the gene of interest should be generated. Many mammalian expression vectors are available commercially and have various purification tags (polyhistidine, hem...

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The 10-layer cell factories are an effective vessel for the production of milligram quantities of protein. A major advantage of using the cell factory over other traditional vessels, such as roller bottles, shake flasks or spinner flasks, is that they do not require the purchase of any additional laboratory equipment. A standard CO2 incubator (~6.0 cu. ft.) will easily accommodate four 10-layer cell factories (Fig. 2). In addition, these vessels require less labor and space than dishes, flasks...

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company </td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Alkaline phosphatase (BCIP/NBT) liquid substrate solution</td>
 <td>Sigma</td>
 <td>B6404</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Antibiotic/Antimycotic, 100X</td>
 <td>Invitrogen</td>
 <td>15240062</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-HA affinity matrix, clone 3F10</td>
 <td>Roche</td>
 <td>1815016</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-HA murine mAb, clone 16B12</td>
 <td>Covance</td>
 <td>MMS-101P</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell culture flask, T75 cm2 tissue culture treated</td>
 <td>Corning</td>
 <td>430641</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell culture flask, T225 cm2 tissue culture treated</td>
 <td>Corning</td>
 <td>431082</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell culture plates,6-well tissue culture treated</td>
 <td>Corning</td>
 <td>3516</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell factory, 10-layer CellSTACK</td>
 <td>Corning</td>
 <td>3312</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Centramate Omega 5K Cassette</td>
 <td>Pall</td>
 <td>OS005C12</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Centramate Omega 30K Cassette</td>
 <td>Pall</td>
 <td>OS030C12</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chromatography glass column, 1.0x10 cm</td>
 <td>Kontes</td>
 <td>4204001010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ciprofloxacin</td>
 <td>Sigma</td>
 <td>17850</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CO2</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dulbecco's modified Eagle's media (DMEM)</td>
 <td>Sigma</td>
 <td>D5796</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal bovine serum (FBS), heat inactivated</td>
 <td>Invitrogen</td>
 <td>12484-028</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FuGENE HD transfection reagent</td>
 <td>Promega</td>
 <td>4709691001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GeneJuice transfection reagent</td>
 <td>EMD/Merck</td>
 <td>70967-6</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glycine</td>
 <td>Sigma</td>
 <td>G8898</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Goat anti-mouse IgG F(ab')2 alkaline</td>
 <td>Thermo Scientific</td>
 <td>31324</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>phosphatase-conjugated antibody</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hemagglutinin (HA) peptide, 100 mg</td>
 <td>Genscript</td>
 <td>custom synthesis</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>(sequence: YPYDVPDYA; 95% purity)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEK 293T cells</td>
 <td>ATCC</td>
 <td>CRL-11268</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Household bleach (4% w/v sodium hypochlorite)</td>
 <td>various brands are available</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Immobilon-P PVDF membrane</td>
 <td>Millipore</td>
 <td>IPVH07850</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MiniPrep plasmid purification kit, PureLink Quick</td>
 <td>Invitrogen</td>
 <td>K2100-11</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MaxiPrep plasmid purification kit, PureLink HiPure</td>
 <td>Invitrogen</td>
 <td>K2100-07</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NaN3</td>
 <td>Sigma</td>
 <td>S8032</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pDISPLAY expression vector</td>
 <td>Invitrogen</td>
 <td>V660-20</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin/streptomycin (pen/strep), 100X</td>
 <td>Invitrogen</td>
 <td>15140-122</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphate-buffered saline (PBS), sterile 1X</td>
 <td>Sigma</td>
 <td>D8537</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polyethyleneimine (PEI), linear 25 kDa</td>
 <td>Polyscience</td>
 <td>23966</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Skim milk dry powder</td>
 <td>Carnation</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Stericup-GP PES vacuum filtration unit,</td>
 <td>Millipore</td>
 <td>SCGPU05RE</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.22 &mu;m, 500 ml capacity</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypan blue</td>
 <td>Invitrogen</td>
 <td>15250061</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypsin-EDTA, 0.05% (w/v)</td>
 <td>Invitrogen</td>
 <td>25300-054</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tween-20</td>
 <td>Sigma</td>
 <td>P7949</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Valproic acid</td>
 <td>Sigma</td>
 <td>P4543</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Centramate tangential flow system</td>
 <td>Pall</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CO2 humidified incubator, standard 6.0 cu. ft.</td>
 <td>various brands are available</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Electrophoresis and transfer unit</td>
 <td>various brands are available</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Incubator, 37 &deg;C</td>
 <td>various brands are available</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

a convenient and general expression platform for the production of secreted proteins from human cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

Watch this Scientific Journal Video about A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells at JoVE.com

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

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21.0K ビュー.  University of Toronto. ポストヒトゲノム時代において、ネイティブコンフォメーションにおける組換えタンパク質の可用性は、構造、機能および治療の研究開発に不可欠である。ここでは、テストと組換えタンパク質のさまざまなを生成するために使用することができるヒト胚性腎臓293T細胞における大規模タンパク質発現系を記述します。

ビデオ: A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant...

bacterial expression and purification of human matrix metalloproteinase-3 using affinity chromatography

Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

His-tag purification, dialysis, and activation are employed to increase yields of soluble, active matrix metalloproteinase-3 catalytic domain protein expression in bacteria. Protein fractions are analyzed via SDS-PAGE...

modifying baculovirus expression vectors to produce secreted plant proteins in insect cells

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect...

production of recombinant prmt proteins using the baculovirus expression vector system

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

The baculovirus expression vector system (BEVS) is a robust platform for expression screening and production of protein arginine methyltransferases (PRMTs) to be used for biochemical, biophysical, and structural studies. Milligram quantities of material can be produced for the majority of PRMTs and other proteins of interest requiring a eukaryotic expression platform.

apoplast-extraction based method to improve the purity of plant produced recombinant protein  

Author Spotlight: Optimizing Apoplast Protein Extraction for Efficient Recovery of Recombinant Proteins from Plant Cells

The purification of recombinant proteins from plant systems is usually challenged by plant proteins. This work provides a method to effectively extract and purify a secreted recombinant protein from the apoplast of Nicotiana...

Efficient Extraction and Purification of GFP-His from &lt;i&gt;N. benthamiana&lt;/i&gt; Leaves

https://cloudfront.jove.com/CDNSource/teasers/66852_b.jpg

label-free imaging of single proteins secreted from living cells via iscat microscopy

Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy

We present a protocol for the real-time optical detection of single unlabeled proteins as they are secreted from living cells. This is based on interferometric scattering (iSCAT) microscopy, which can be applied to a variety of different biological systems and...

identification of the source of secreted proteins in the kidney by brefeldin a injection

Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection

Identifying the cell type responsible for secreting cytokines is necessary to understand the pathobiology of kidney disease. Here, we describe a method to quantitatively stain kidney tissue for cytokines produced by kidney epithelial or interstitial cells using brefeldin A, a secretion inhibitor, and cell-type-specific markers.

efficient and scalable production of full-length human huntingtin variants in mammalian cells using a transient expression system

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

We provide scalable protocols covering construct design, transient transfection, and expression and purification of full-length human huntingtin protein variants in HEK293...

expression of functional recombinant hemagglutinin and neuraminidase proteins from the novel h7n9 influenza virus using the baculovirus expression system

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface...

high yield expression of recombinant human proteins with the transient transfection of hek293 cells in suspension

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan...

production and characterization of human macrophages from pluripotent stem cells

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived...

the multibac protein complex production platform at the embl

The MultiBac Protein Complex Production Platform at the EMBL

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its...

a multilayer microfluidic platform for the conduction of prolonged cell-free gene expression

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is...

serum free production of three-dimensional human hepatospheres from pluripotent stem cells

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical...

optimized protocol for the extraction of proteins from the human mitral valve

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve...

production of lentiviral vectors for transducing cells from the central nervous system

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

In this protocol we describe production, purification and titration of lentiviral vectors.  We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes.  Our methods may also apply to other cell types in vitro and in...

the production of pluripotent stem cells from mouse amniotic fluid cells using a transposon system

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

a high yield and cost-efficient expression system of human granzymes in mammalian cells

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

reverse phase protein arrays based protein expression analysis: a procedure for simultaneous quantification of expression of multiple proteins from cell lysate

This video demonstrates the methodology for performing Reverse Phase Protein Arrays (RPPA) to study protein expression patterns from cell lysate.&#160;In RPPA, the lysate containing a mixture of proteins is printed on nitrocellulose slides and proteins of interest are analyzed using fluorescently labeled...

Reverse Phase Protein Arrays Based Protein Expression Analysis: A Procedure for Simultaneous Quantification of Expression of Multiple Proteins from Cell Lysate

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...